1. The Binding of κ-Conotoxin PVIIA and Fast C-Type Inactivation of Shaker K+ Channels are Mutually Exclusive 
E. Dietlind Koch, Baldomero M. Olivera, Heinrich Terlau, Franco Conti 
Biophysical Journal 
Volume 86, Issue 1, Pages (January 2004)
DOI: /S (04)
Copyright © 2004 The Biophysical Society Terms and Conditions

2. Figure 1
Effect of κ-PVIIA on the potassium currents mediated by wild-type and mutant Shaker channels. Each panel shows the superposition of responses to 0mV steps obtained from the same oocyte before and after the external addition of κ-PVIIA at concentrations of 100 nM (panels 1 and 3) or 250 nM (panels 2 and 4). For better comparison currents are adjusted to show control records with similar peak amplitudes at two different timescales. (From left to right) Wild-type Shaker channels (Shaker); channels deprived of fast inactivation by the 6-46 N-terminal deletion (Shaker-Δ6-46); channels with the additional point mutation M448K (Δ6-46-M448K); or with the additional T449S mutation (Δ6-46-T449S). (Upper panel) Notice that the toxin slows drastically the relatively fast C-type inactivation of M448K and T449S, but has no similar effect on either the N-type inactivation of WT-Shaker or the C-type inactivation of Shaker-Δ6-46. (Lower panel) At an expanded timescale it becomes obvious that the apparent effects on the activation are similar for all phenotypes.
Biophysical Journal  , DOI: ( /S (04) )
Copyright © 2004 The Biophysical Society Terms and Conditions

3. Figure 2
Steady-state binding of κ-PVIIA to the closed state of M448K and T449S channels. (A) The solid lines show the rising phase of M448K currents elicited by steps to 0, +20, and +40mV from a holding potential of −100mV under control conditions; the solid dots are sampled points of similar responses after the addition of 100 nM κ-PVIIA, scaled by a factor of 2.5 to make them match the early rise of the control records. The dashed lines are equally scaled fits of the toxin records with Eq. 1, obtained for a fixed U0=0.41 and for R-values increasing from 13s−1 at 0mV to 38s−1 at 40mV. (B) Dependence of U0 on κ-PVIIA concentration, [T]. The solid line represents a fit using U0([T])=1/(1+[T]/Kc) resulting in a dissociation constant for κ-PVIIA binding of 69 nM (see text). (C) Data of the same type as in A for T449S-mediated currents under control conditions and after addition of 300 nM κ-PVIIA. The dashed lines fitting toxin data to Eq. 1 were obtained for a fixed U0=0.42 and for R-values increasing from 95s−1 at 0mV to 290s−1 at 40mV. (D) Dependence of U0 on toxin concentration. The fit (solid line) corresponds to a dissociation constant for κ-PVIIA binding of 223 nM.
Biophysical Journal  , DOI: ( /S (04) )
Copyright © 2004 The Biophysical Society Terms and Conditions

4. Figure 3
Relaxation of κ-PVIIA binding to closed M448K channels revealed by double-pulse stimulation. (A) Currents elicited by double pulses to +40mV with variable interpulse interval, Ti, from the holding potential of −100mV under control conditions. The currents elicited by the first pulse, I1, are all superimposed; the responses to the second pulse, I2, for the indicated interpulses, are displayed with an arbitrary horizontal shift. (B) Normalized amplitude of the second response as a function of Ti for the data shown under A (solid circles). The single exponential fit corresponds to a rate constant for recovery from C-type inactivation, λI ≈ 1.41s−1 (smooth line). (C) Currents measured from the same oocyte as in A using the same pulse protocol after addition of 100 nM κ-PVIIA. Notice the overshoot and the different inactivation kinetics of the currents elicited by the second pulse as compared to the first. (D) The fraction of active and unblocked channels, U0, measured from the second response under toxin by comparison with the first control response as in Fig. 2 A, is plotted as a function of Ti (solid circles). The smooth line is the best fit of the data according to Eq. 2 (see text). (E) Direct comparison of sampled current traces in control (continuous lines) and in 100 nM κ-PVIIA (dotted lines). Notice that after an interpulse period of 256ms the response elicited by the second pulse in the two different conditions is almost identical; only for longer Ti the second response under toxin acquires progressively the smaller amplitude and the slower inactivation that characterize κ-PVIIA binding to the channels.
Biophysical Journal  , DOI: ( /S (04) )
Copyright © 2004 The Biophysical Society Terms and Conditions

5. Figure 4
The binding relaxation of κ-PVIIA to the closed state of M448K channels during double-pulse stimulations results from a bimolecular binding reaction. (A) Relative fractions of P2 responses as a function of Ti measured in the absence (Control) and in the presence of 100 nM (solid circles) or 300 nM (unfilled squares) κ-PVIIA. The double-pulse protocol consisted of 160-ms pulses with Ti between 16ms and 8s from a holding potential of −100mV. For the control currents the smooth line corresponds to a single exponential fit resulting in a rate constant for recovery from C-type inactivation of λI=1.4s−1. The smooth lines for the toxin traces are obtained by using Eq. 2 and correspond to on=0.69s−1, and off=0.35s−1 for 100 nM κ-PVIIA and on=2.5s−1 and off=0.42s−1 for 300 nM κ-PVIIA. (B) Concentration dependence of on and off binding-rates of κ-PVIIA to closed M448K channels. The straight lines are fits of the data according to off=koff=5.33s−1 and on=kon×T, kon=0.41μM−1 s−1.
Biophysical Journal  , DOI: ( /S (04) )
Copyright © 2004 The Biophysical Society Terms and Conditions

6. Figure 5
Recovery of tonic κ-PVIIA binding to closed T449S channels. (A) Superimposed records of P2 responses from double pulses (Vp=40mV, Tp=4ms) with variable pulse interval, Ti, in the presence of 150 nM κ-PVIIA. With increasing Ti the current onset of the P2 responses becomes slower, indicating an increasing fraction of initially blocked channels. The P2 response for Ti=1s was almost identical to the P1 response (not shown) which showed an early unblock probability, Uc=0.58 by comparison with control. (B) Relative charge (QP2/QP1) as a function of interpulse interval in the absence of toxin (unfilled circles) and in the presence of 150 nM κ-PVIIA (solid circles). The solid line is a single-exponential decay with τ=260ms.
Biophysical Journal  , DOI: ( /S (04) )
Copyright © 2004 The Biophysical Society Terms and Conditions

7. Figure 6
Concentration and voltage-dependence of κ-PVIIA binding relaxations to open M448K channels. (A) Effect of κ-PVIIA on M448K currents elicited by +40mV pulses. Current traces resulting from pulses to +40mV in the absence (Control) and presence of 300 nM, 1μM, or 3μM κ-PVIIA in the external solution. Increasing toxin concentration leads to a reduction in peak currents and to an increased slowing of the inactivation. The solid lines, almost indistinguishable from raw data, are either a monoexponential fit of the inactivation decay for t>2×tpeak (Control), or double-exponential fits of the biphasic time course of toxin records in the same time interval. The time constants for the late inactivation are: Control, 11.7ms; 300 nM κ-PVIIA, 21.3ms; 1μM κ-PVIIA, 26.7ms; and 3μM κ-PVIIA, 40.3ms. The dashed line corresponds to 0 currents. (B) M448K-mediated currents upon stimulation to 0, +20, +40, and +60mV from a holding potential of −100mV in the presence of 1μM κ-PVIIA. Notice the increase in the rate of inactivation at higher test voltages. Data are from the same oocyte of Fig. 6 A. The dashed line corresponds to 0 currents. (C) Voltage-dependence of the rate-constants characterizing inactivation and κ-PVIIA binding to open M448K channels. (Unfilled triangles) Inactivation rate of control currents, λh×s. (Unfilled squares) Second-order association rate-constant, kon×μM×s. (Solid squares) First-order dissociation rate-constant, koff×s. The kon and koff estimates were obtained by unfolding toxin-binding relaxations from inactivation as described in the text. λh and kon data are connected by simple line segments. The thicker line through koff data is a least-squares fit with the function koff (V)=koff(0)×exp(V/vs). The kon data do not show any systematic voltage-dependence and have a mean value of 42μM−1 s−1.
Biophysical Journal  , DOI: ( /S (04) )
Copyright © 2004 The Biophysical Society Terms and Conditions

8. Figure 7
Dose-dependence of the effect of κ-PVIIA on T449S currents. (A) Current responses elicited by step-depolarizations to +40mV in toxin-free conditions (Control) and in presence of 1μM or 3μM κ-PVIIA in the external solution. Increasing toxin concentration, [T], leads to a progressive reduction in peak currents and slowing of the inactivation. The decay phase of the control record is fitted by a double-exponential function with slow and fast rates, λs=12.2s−1 and λf=111s−1; the fitted rates of the dominant slow component of the toxin records are 10.6s−1 for [T]=1μM and 8.1s−1 for [T]=3μM. (B) Estimates of the unblock probability of activated channels at 40mV, U(A), derived from the decrease of λs values (see text), are plotted as a function of [T]. The solid line is the best fit of the data with the function U(A)=K(A)/(K(A)+[T]) with an apparent dissociation constant K(A)=3.95μM and n=14 at 0.3, 10 at 1, 6 at 3, and 3 at 10M. (C) Comparison of the early phases of the current responses shown in A; the delayed onset of the currents in the presence of 1 and 3μM κ-PVIIA is fitted by the convolution of control activation and toxin-binding relaxation according to Eq. 4 (see text). The fitted binding-relaxation rates are: 1μM, λB=610s−1 and 3μM, λB=820s−1. (D) [T]-dependence of estimated toxin-binding kinetics at +40mV. (Unfilled circles) λB estimates from the delay of early currents. (Solid circles) The off rate estimated as the product of λB×the unblock probability, U(A), derived from the slowing of inactivation as in A and B. (Unfilled squares) The on rates estimated as λB×(1-U(A)). The straight lines are best fits according to λB=koff+kon×[T], with off=koff, on=kon×[T], koff×s=600±40s−1, and kon=140±30μM−1 s−1. Data is given as mean±SD; n=14 at 300 nM, 10 at 1μM, and 6 at 3μM.
Biophysical Journal  , DOI: ( /S (04) )
Copyright © 2004 The Biophysical Society Terms and Conditions

9. Figure 8
Voltage-dependence of κ-PVIIA binding to open T449S channels. (A) T449S-mediated currents upon stimulation to +20 and +60mV from a holding potential of −100mV in the absence and presence of 10μM κ-PVIIA. The inactivation kinetics in control conditions are fairly voltage-independent (20mV, λs=11.9s−1 and 60mV, λs=12.2s−1), whereas the slowing of inactivation by κ-PVIIA is much more pronounced at lower voltages (20mV, λs=6, 8s−1 and 60mV, λs=9.7s−1). (B) Voltage-dependence of the dissociation constant of κ-PVIIA binding to open T449S channels, estimated as in Fig 7Fig 7 A. The solid line is the best fit of the data to K(A)=K(A)(0)×exp (V/vs) with K(A)(0)=1.3 (±0.05) μM and vs=36 (±2) mV (n=33). (C) Early phase of the currents in A on an expanded timescale. Notice that the delay in the currents elicited in the presence of κ-PVIIA is strongly decreased at higher test potentials, indicating a faster unbinding of the toxin. (D) Estimates of koff and kon, obtained as described in Fig. 7 D, are plotted against test potential. The solid line through koff data is the best fit to koff=koff(0)×exp (V/vs) with koff(0)=195 (±10) s−1 and vs=38 (±2) mV. The straight line through kon data corresponds to kon=133 (±5) μM−1 s−1. Data are given as mean±SD; the n for the different test voltages was between 15 and 33.
Biophysical Journal  , DOI: ( /S (04) )
Copyright © 2004 The Biophysical Society Terms and Conditions

10. Figure 9
κ-PVIIA does not decrease Shaker-M448K-mediated currents. (A) M448K-mediated currents and the corresponding charge values evoked by stimulations to +40mV from a holding potential of −100mV before (Control) and after addition of 300 nM κ-PVIIA. (B) Plot of the charge against test potential for Vp≥0. (C) Ratio of toxin to control charge values for Vp≥0 as a function of toxin concentration. The presence of κ-PVIIA leads to a charge increase of ∼12% at higher toxin concentrations. The solid line corresponds to a dose response curve with an IC50 of 72 nM. Data are given as mean±SE; the n for the different toxin concentrations was between 10 and 43.
Biophysical Journal  , DOI: ( /S (04) )
Copyright © 2004 The Biophysical Society Terms and Conditions

11. Figure 10
κ-PVIIA prevents cumulative C-type inactivation and shifts steady-state inactivation. (A) M448K-mediated currents elicited by a pulse to +40mV after a variable conditioning period at −30mV, in the absence (Control) or in the presence of 500 nM κ-PVIIA. The holding potential was −100mV. (B) Comparison of the currents shown in A for no conditioning (first pulse) and after a conditioning period of 4s. Notice the increase in current in the presence of toxin. (C) Steady-state inactivation curve of M448K channels without (Control) and with 1μM κ-PVIIA in the bath solution. Normalized peak currents are plotted against prepulse potential. Cells were depolarized to +40mV after being held for 60s at different prepulse potential. Data are given as mean±SE; n=3. The solid lines correspond to a Boltzmann fit indicating that the presence of 1μM κ-PVIIA leads to a shift in the V1/2 for inactivation of 6.4mV.
Biophysical Journal  , DOI: ( /S (04) )
Copyright © 2004 The Biophysical Society Terms and Conditions

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